1. Field of the Invention
The present invention relates generally to the field of viral vectors, packaging cell lines, and the use of such viral vectors to express foreign DNA in mammalian cells. The invention also relates to the field of gene therapy, and more specifically to the use of viral vectors to transport genetic material into cells in vivo for therapeutic purposes. More particularly, it concerns viral promoter replacement in order to reduce the expression levels of viral genes in host cells.
2. Description of the Related Art
Gene therapy is an area that offers an attractive approach for the treatment of many diseases and disorders. Many diseases are the result of genetic abnormalities such as gene mutations or deletions, and thus the prospect of replacing a damaged or missing gene with a fully functional gene is provocative. Throughout the last decade, studies of oncogenes and tumor suppressor genes have revealed increasing amounts of evidence that cancer is a disease caused by multiple genetic changes (Chiao et al., 1990; Levine, 1990; Weinberg, 1991; Sugimara et al., 1992). Based on this concept of carcinogenesis, new strategies of therapy have evolved rapidly as alternatives to conventional therapies such as chemo- and radiotherapy (Renan, 1990; Lotze et al., 1992; Pardoll, 1992). One of these strategies is gene therapy, in which tumor suppressor genes, antisense oligonucleotides, and other related genes are used to suppress the growth of malignant cells.
Gene therapy has also been contemplated for transfer of other therapeutically important genes into cells to correct genetic defects. Such genetic defects include deficiencies of adenosine deaminase that result in severe combined immunodeficiency, human blood clotting factor IX in hemophilia B, the dystrophin gene in Duchenne muscular dystrophy, and the cystic fibrosis transmembrane receptor in cystic fibrosis. Gene transfer in these situations requires long term expression of the transgene, and the ability to transfer large DNA fragments, such as the dystrophin cDNA, which is about 14 kB in size.
High efficiency transduction of cells and the ability to administer multiple doses of a therapeutic gene are particularly important points in gene therapy. The ability to transfer a gene into a cell requires a method of transferring the new genetic material across the plasma membrane of the cell and subsequent expression of the gene product to produce an effect on the cell. There are several means to transfer genetic material into a cell, including direct injection, lipofection, transfection of a plasmid, or transduction by a viral vector. The natural ability of viruses to infect a cell and direct gene expression make viral vectors attractive as gene transfer vectors. Other desirable elements of gene transfer vectors include a high transduction efficiency, large capacity for genetic material, targeted gene delivery, tissue-specific gene expression, and the ability to minimize host immunologic responses against the vector.
One particularly gene therapy vector, adenovirus, has been widely studied and well-characterized as a model system for eukaryotic gene expression, and have become the vector of choice for in vivo gene transfer. Adenoviruses are easy to grow and manipulate, and they exhibit broad host range both in vitro and in vivo. They can be produced to high titers, e.g., 109-1011 plaque-forming units (PFU)/ml, and they are highly infectious for both dividing and non-dividing cells. The life cycle of adenovirus does not require integration into the host cell genome; the foreign genes encoded by adenovirus vectors are expressed episomally, and therefore have low genotoxicity to the host cells. Adenoviruses are not, however, associated with any significant pathologies. They appear only to be linked to mild forms of disease, and there are no known human malignancies associated with adenovirus infection. Moreover, no side effects have been reported in studies of vaccination with wild-type adenovirus (Couch et al., 1963; Top et al., 1971), demonstrating their safety and therapeutic potential as in vivo gene transfer vectors.
Many viral vectors have not showed the in vivo results that many have hoped, adenovirus being one of these. Expression levels and duration of expression appear to be two problems. It is thought that one of the causes for these problems is the toxicity and immunogenicity of adenovirus, especially and high dosage.
One way of attaining this goal is to reduce or eliminate the expression of adenoviral proteins in the host. The diminution of viral gene expression and viral replication is desirable for the development of viral vectors used for gene therapy, for attenuated live viral vaccines and for the transformation of cells in vitro for the purpose of protein production. A common approach to this endeavor in the adenoviral system has been to delete certain viral genes. Of course, if the gene is essential to viral replication, the function must be complemented. This complementation is accomplished by providing a xe2x80x9chelperxe2x80x9d cell line that is transformed with a copy of the deleted viral gene. When this cell is infected, the gene produces its essential product, thereby allowing the virus to replicate. However, in a cell not so transformed, the virus can infect but will not replicate.
Thus, there are many benefits to be obtained by the development of new viral vectors and methods for reducing the viral gene expression of such vectors in host cells.
The present invention overcomes deficiencies in the prior art by providing new viral vectors and methods for reducing the viral gene expression of such vectors in host cells.
More particularly, the present invention provides a viral vector containing at least one essential viral gene or gene element under the control of an inducible promoter. In preferred embodiments, the inducible promoter is a yeast GAL4 promoter. In particular embodiments of the present invention, the vector is derived from adenovirus and contains an adenoviral origin of replication. In certain other embodiments, the essential viral gene or gene element is selected from the group consisting of E1A, E1B, E2, E4 and E5. As used herein, the term xe2x80x9cgene elementxe2x80x9d may be defined as any DNA sequence that comprises a promoter element operably linked to a piece of DNA that encodes a polypeptide or protein product.
In certain embodiments, a viral vector is provided, wherein at least one viral gene or gene element is deleted therefrom. In such aspects of the invention, the deleted viral gene or gene element is selected from the group consisting E1A, E1B, E2, E3, E4 and E5, and the essential viral gene or gene element and the deleted viral gene and gene element are different. In certain embodiments, the E2 gene also is under the control of an inducible promoter. In other embodiments, the E5 gene also is under the control of an inducible promoter. In some embodiments, it is contemplated that at least two viral genes or gene elements are deleted. In particular embodiments the deleted genes may be E1A and E1B. In other aspects it is contemplated that the E3 gene also is deleted.
In particular embodiments there is provided a viral vector wherein the essential viral gene or gene element is E4. Other embodiments of the invention provide a viral vector containing at least one essential gene or gene element under the control of an inducible promoter, wherein the inducible promoter is selected from the group consisting of the auxin inducible promoter, tet-responsive element and an ecdysone hybrid response element. It is contemplated that a viral vector may further comprise a heterologous gene. In these aspects the heterologous gene is under the control of a promoter active in eukaryotic cells. In particular embodiments the promoter may be CMV promoter.
In certain embodiments the viral vector of the present invention further comprises a polyadenylation signal in operable relation to the heterologous gene. In particular aspects of the present invention the polyadenylation signal is selected from the group consisting of adenovirus, SV40 and bovine growth hormone. In certain embodiments the heterologous gene is selected from the group consisting of a tumor suppressor, an antisense transcript, a vaccine antigen and a single-chain antibody. In certain embodiments of the present invention the tumor suppressor is selected from the group consisting of but not limited to p53, RB, APC, DCC, NF-1, NF-2, WT-1, MEN-I, MEN-II, BRCA1, VHL, FCC, MCC, MMAC1, zac1, p16, p21; p57, p73, p27, C-CAM and BRAC2. In other embodiments, the antisense transcript may comprise antisense against oncogenes such as, for example, ras, myc, neu, raf erb, src, fms, jun, trk, ret, gsp, hst, bcl and abl. The heterologous gene, in alternative embodiments, may encode an inducer of apoptosis, such as Bax, Bak, Bcl-X, Bik, Bid, Harakiri, Ad E1B, Bad and ICE-CED3 proteases.
Other aspects of the present invention provide a cell comprising a heterologous gene encoding at least a first factor that induces a promoter that is capable of activity in eukaryotic cells. In particular embodiments the first factor is a fusion polypeptide of VP16 and a fusion partner, the term xe2x80x9cfusion partnerxe2x80x9d refers to a polypeptide that may bind to an element within a promoter region. As used herein the term xe2x80x9cfusion proteinxe2x80x9d or xe2x80x9cfusion polypeptidexe2x80x9d is a protein or polypeptide encoded by two fused genes or gene elements. In particular embodiments the fusion partner for VP16 is selected from the group consisting of GAL4, tet repressor, ecdysone receptor and auxin. In other embodiments the factor is a fusion polypeptide of the estrogen receptor hormone binding domain and a fusion partner. In such embodiments, the fusion partner for estrogen receptor hormone binding domain is selected from the group consisting of GAL4, tet repressor/VP16 fusion protein, ecdysone receptor and auxin. In further embodiments the GAL4 fused to VP16.
The cell may further comprise at least one viral gene or gene element essential to the replication of the corresponding virus. In preferred aspects the viral gene or gene element is an adenoviral gene selected from the group consisting of E1A, E1B, E2, E4 and E5. In certain embodiments the adenoviral gene is E1B. In other embodiments the adenoviral gene further comprises the E1A gene. In specific embodiments the adenoviral gene further comprises the E2 gene. In other embodiments it is envisioned that the adenoviral gene is E1A.
In preferred embodiments, the cell further comprises a gene encoding at least at second factor that induces a promoter that is capable of activity in eukaryotic cells. In one embodiment the second factor is a fusion polypeptide of VP16 and a fusion partner. In another embodiment, the second factor is a fusion polypeptide of the estrogen receptor and a fusion partner.
Also provided by the present invention are methods for producing an infectious, conditionally replication-defective viral particles comprising providing a cell comprising a heterologous gene encoding at least a first factor that induces a promoter that is capable of activity in eukaryotic cells, contacting the cell with a viral vector, the viral vector comprising at least one essential viral gene or gene element under the control of a promoter that is induced by the first factor and inactive in the absence of the factor, culturing the cell under conditions permitting the uptake of the viral vector by, and replication in, the cell; and harvesting infectious virus particles produced by the cell.
In specific embodiments the cell further comprises an essential viral gene or gene element and the vector lacks a functional copy of the essential viral gene. In other embodiments the viral vector comprises a heterologous gene. In preferred embodiments the viral vector is derived from adenovirus and contains an adenoviral origin of replication.
The present invention also provides a method for producing a protein in a cell comprising contacting the cell with an infectious viral particle, the particle comprising a viral vector comprising at least one essential viral gene or gene element under the control of a promoter that is induced by the first factor and inactive in the absence of the factor and a heterologous gene, and culturing the cell under conditions permitting the uptake of the particle by the cell and the synthesis of the product of the beterologous gene in the cell, the conditions not including the first factor. In preferred embodiment the method further comprises the step of isolating the product. In other preferred embodiments the viral vector is derived from adenovirus and contains an adenoviral origin of replication.
Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.